Structure and mechanism of CpG specific DNA glycosylases

CpG特异性DNA糖基化酶的结构和机制

• 批准号: 7146414
• 负责人: Alex C Drohat
• 金额: $ 24.47万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7146414
• 关键词: CpG islands; DNA damage; DNA repair; N glycosidase; catalyst; chemical kinetics; endonuclease; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; hydrolysis; intermolecular interaction; ionic bond; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; site directed mutagenesis; thermodynamics; thymine

DESCRIPTION (provided by applicant): The integrity of the genetic information encoded by DNA is essential to all organisms, yet the reactive bases of DNA are continuously subjected to chemical modification from endogenous and exogenous sources. To counteract this inevitable damage, the cellular machinery includes DNA repair systems. Damaged bases in DNA are repaired via base excision repair (BER), initiated by a damage-specific DNA glycosylase. These enzymes find lesions within the vast genomic DNA, and hydrolyze a generally stable bond to release the damaged base, producing apurinic/apyrimidinic (AP) DNA. DNA glycosylases typically bind the cytotoxic AP DNA product tightly until displaced by an AP endonuclease to continue BER. Although some enzymes recognize a single lesion; e.g., uracil DNA glycosylase is exquisitely specific for uracil, others recognize multiple lesions and/or mismatched bases. Two human enzymes recognize G/T mispairs, and other mutagenic lesions, specifically at CpG sites; methyl-binding domain IV (MBD4) and thymine DNA glycosylase (TDG). The long-term goal of this research is to understand how these enzymes recognize complex and multiple forms of damage and yet exclude normal base pairs, how they catalyze the hydrolysis of a generally stable bond, and how the AP DNA product is transferred from the DNA glycosylase to the AP endonuclease. The focus of this proposal is to determine how TDG obtains its specificity for G/T mispairs and other lesions, and its specificity against normal GC pairs, and how human AP endonuclease (APE1) stimulates the release of AP DNA from TDG. Towards this end, we will employ a multidisciplinary approach including structural, biophysical, and biochemical methods. The specific aims are to (i) determine the NMR structure of the TDG catalytic domain (TDGc), (ii) characterize the TDG reaction mechanism using transient and steady-state kinetics, and equilibrium binding experiments, (iii) discover the chemical basis of the recognition of multiple substrates and the rejection of GC by TDG, (iv) elucidate the mechanism of AP DNA transfer between TDG and APE1, and (v) determine the NMR structure of a binary TDGc-DNA substrate analog complex to reveal the structural basis of TDG specificity. Given the mutagenic and cytotoxic effects of damage occurring at CpG sites in human genomic DNA, the proposed structural and mechanistic studies of TDG may have significant implications for ageing, and diseases including cancer.

描述（由申请人提供）：DNA 编码的遗传信息的完整性对于所有生物体至关重要，但 DNA 的反应碱基不断受到内源和外源的化学修饰。为了抵消这种不可避免的损伤，细胞机制包括 DNA 修复系统。 DNA 中受损的碱基通过碱基切除修复 (BER) 进行修复，该修复由损伤特异性 DNA 糖基化酶启动。这些酶发现大量基因组 DNA 中的损伤，并水解通常稳定的键以释放受损的碱基，产生无嘌呤/无嘧啶 (AP) DNA。 DNA 糖基化酶通常紧密结合细胞毒性 AP DNA 产物，直到被 AP 核酸内切酶取代以继续 BER。尽管有些酶可以识别单个病变；例如，尿嘧啶 DNA 糖基化酶对尿嘧啶具有高度特异性，其他酶可识别多个损伤和/或错配碱基。两种人类酶可识别 G/T 错配和其他诱变病变，特别是在 CpG 位点；甲基结合域 IV (MBD4) 和胸腺嘧啶 DNA 糖基化酶 (TDG)。这项研究的长期目标是了解这些酶如何识别复杂和多种形式的损伤，但排除正常碱基对，它们如何催化一般稳定键的水解，以及 AP DNA 产物如何从 DNA 转移糖基化酶转化为AP核酸内切酶。该提案的重点是确定 TDG 如何获得对 G/T 错配和其他病变的特异性，以及对正常 GC 对的特异性，以及人 AP 核酸内切酶 (APE1) 如何刺激 TDG 释放 AP DNA。为此，我们将采用多学科方法，包括结构、生物物理和生物化学方法。具体目标是 (i) 确定 TDG 催化结构域 (TDGc) 的 NMR 结构，(ii) 使用瞬态和稳态动力学以及平衡结合实验表征 TDG 反应机制，(iii) 发现 TDG 催化结构域 (TDGc) 的化学基础TDG 对多种底物的识别和 GC 的排斥，(iv) 阐明 TDG 和 APE1 之间 AP DNA 转移的机制，以及 (v) 确定二元 TDGc-DNA 的 NMR 结构底物类似物复合物揭示了 TDG 特异性的结构基础。鉴于人类基因组 DNA 中 CpG 位点发生损伤的诱变和细胞毒性作用，拟议的 TDG 结构和机制研究可能对衰老和包括癌症在内的疾病具有重大影响。